The great majority of HIV-1 strains enter CD4+ target cells by interacting with one of two coreceptors, CCR5 and CXCR4. It has been considered that alternative coreceptors are not been used for primary clinical infection. However, we recently discovered a transmitted/founder (T/F) virus (ZP6248) that was profoundly impaired in its ability to utilize CCR5 and CXCR4 coreceptors on multiple CD4+ cell lines as well as primary human CD4+ T cells and macrophages in vitro, yet replicated to a high plasma viral load during acute infection. In contrast to all other T/F viruses that invariably use CCR5 as a coreceptor, ZP6248 uses the coreceptor GPR15 most efficiently in in vitro assays. A single mutation (E314G) in the rare V3 crown tip of ZP6248 restored its infectivity in CCR5+ cells, but reduced its ability to replicate in GPR15+ cells. Introduction of glutamic acid at this same position in other virus backbones leads to significant alterations of coreceptor usage, but does not render them infectious in GPR15+ cells. This is the first case demonstrating that HIV-1 can use coreceptors other than CCR5 and CXCR4 to establish clinical infection. Thus, it will be important to understand what are the mechanisms involved in establishing a primary HIV-1 infection in humans without the use of CCR5 or CXCR4 as coreceptors. In addition, it is striking that a single amino acid substitution at the V3 crown tip leads to significantly altered coreceptor usage in different virus backbones. These findings provide a unique model to study the precise interactions between the V3 crown tip and various coreceptors. Thus, we will use this unique virus as a model to address the following fundamental biological questions that are critical for understanding of coreceptor tropism of this evolving viral pathogen: (i), what novel infection mechanisms are used by a transmitted HIV-1 variant with atypical coreceptor usage and what are the motifs in the envelope glycoprotein that govern the coreceptor tropism; (ii), how conformational changes caused by a single amino acid substitution in the V3 crown tip allows the virus to sample alternative coreceptors to establish clinical infection. This work is central t our understanding and anticipation of virus-host coevolution that will drive the future of the HIV-1 epidemic.